Resistance to chemotherapy is a major obstacle to successful cancer treatment, and it often accounts for the failure of aggressive chemotherapy to eradicate malignant disease(1) . Most metastatic cancers are either innately resistant to chemotherapy or acquire drug resistance during the course of chemotherapy(1) . Multidrug resistant (MDR) ()cancer cells are characterized by broad spectrum resistance to chemotherapeutic drugs, markedly reduced intracellular accumulation of the drugs, and overexpression of the drug efflux pump P-glycoprotein(1, 2) . The relevance of MDR to clinical drug resistance in cancer therapy is indicated by the abundant expression of P-glycoprotein and its message mdr1 in specimens of human cancer that are intrinsically resistant to chemotherapy and in malignant tumors from patients who have relapsed during or after chemotherapy(1, 3, 4) .

Protein kinase C (PKC) is an isozyme family with at least ten mammalian members(5) . Highly selective phorbol-ester PKC activators induce resistance in cancer cells to multiple cytotoxic drugs that are P-glycoprotein substrates in association with a sharp reduction in the intracellular accumulation of the drugs, providing evidence that PKC activation contributes to MDR. The phorbol-ester effects on chemosensitivity and drug accumulation have been observed in several drug-sensitive and MDR cancer cell lines including human breast cancer MCF7-WT and MCF7-MDR, and the magnitude of the effect is generally a 2-6-fold increase in the IC values of cytotoxic drugs (6, 7, 8, 9, 10, 11, 12) . PKC phosphorylates the linker region of P-glycoprotein in MDR human KB-V1 cancer cells(9, 13, 14) , and this is tightly coupled to the effects of PKC on intracellular drug accumulation and MDR, providing evidence that PKC regulates the function of P-glycoprotein in this system(11) . The isozyme PKC- has been shown to be overexpressed in several MDR cancer cell lines including MCF7-MDR(8, 15, 16, 17, 18) . Evidence that PKC- activation contributes to MDR has been provided by observations that MDR is induced by the selective activation of PKC- in human colon cancer cells (12) and by transfection of an mdr1-transfected human breast cancer MCF7 subline with PKC-(19) .

Verapamil, cyclosporin A, and related MDR reversal agents that compete with chemotherapeutic drugs for binding sites on P-glycoprotein (20, 21, 22, 23) generally cause severe toxicity in vivo at therapeutic concentrations, and this precludes their use in the treatment of drug-resistant cancer(24, 25) . In a previous report, we characterized the mechanism of PKC inhibition by an N-myristoylated peptide corresponding to the autoinhibitory pseudosubstrate sequence of PKC- (P1) that had been shown to selectively inhibit PKC in human fibroblasts(26, 27) . In this report, we show that the peptide P1 reverses MDR by a novel mechanism that is associated with a sharp increase in intracellular drug accumulation and inhibition of the phosphorylation of P-glycoprotein and two other PKC- substrates, Raf-1 kinase and PKC- itself. P1 was distinguished from verapamil and related MDR reversal agents(20, 21, 22, 23) in that it did not inhibit photoaffinity labeling of drug-binding sites on P-glycoprotein by [H]azidopine. P1 did not affect P-glycoprotein expression in MCF7-MDR cells. MCF7-MDR cells were not cross-resistant to P1, providing indirect evidence that the peptide was not transported by P-glycoprotein. The N-myristoylated PKC- pseudosubstrate peptide P1 may be a valuable starting point for developing a new generation of MDR reversal agents that function by a novel mechanism that involves inhibition of PKC--catalyzed P-glycoprotein phosphorylation.

MATERIALS AND METHODS

Cell Lines

Synthetic Peptides

Chemicals and Reagents

Drug Accumulation and Efflux Assays

Growth Inhibition Assay

P-glycoprotein, Raf-1 Kinase, and PKC- Phosphorylation in MCF7 Cells

The procedures used to immunoprecipitate P-glycoprotein, PKC-, and Raf-1 kinase were based on previously described methods(18) . For P-glycoprotein immunoprecipitation, the supernatant (100 µg of protein) was incubated with 5 µg of C219 monoclonal antibody by rotation for 16 h at 4 °C. In the case of Raf-1 kinase and PKC- immunoprecipitation, the supernatant was incubated with Raf-1 kinase mAb (5 µg) (Oncogene Science, Uniondale, NY) or PKC- mAb (5 µg) (Upstate Biotechnology, Inc., Lake Placid, NY) by rotation for 2 h at 4 °C, and rotation was continued in the presence of 5 µg rabbit antimouse IgG for an additional 30 min. at 4 °C. Next, 0.2 ml of a 25% (v/v) suspension of protein A-Sepharose was added to the incubation mixture followed by rotation for 30 min at 4 °C. After a 1-min centrifugation in a microcentrifuge, the beads were recovered as a pellet, and they were washed successively with 1 M NaCl, 1% Nonidet P-40, and lysis buffer containing 1 M urea. To recover immune complexes from the beads, the beads were incubated for 10 min at 30 °C with 0.1 ml of SDS-PAGE sample buffer, and then they were pelleted in a microcentrifuge. The supernatant was recovered and subjected to SDS-PAGE. Gels were silver stained, and autoradiography was done.

P-glycoprotein and Raf-1 Kinase Expression

Photoaffinity labeling of P-glycoprotein in MCF7-MDR cells

Statistical Analysis

RESULTS

Table 1shows the structures of the peptides under investigation for modulatory effects on the intracellular accumulation and growth-inhibitory activity of cytotoxic anticancer drugs in human breast cancer MCF7-MDR cells. P2 contains the core sequence of the pseudosubstrate region of PKC- (PKC[20-28]), P4 contains a more extensive PKC- pseudosubstrate sequence (PKC[19-31])(26, 27) , and P6 contains the sequence of the PKC phosphorylation site of the epidermal growth factor receptor at Thr(27) . P1, P3, and P5 are N-myristoylated analogs of P2, P4, and P6, respectively. Each of these N-myristoylated peptides inhibits the histone kinase reaction of purified PKC (Table 1); the corresponding nonmyristoylated peptides do not inhibit PKC-catalyzed histone phosphorylation (Table 1). P7 is an N-myristoylated peptide with a sequence that corresponds to a PKC phosphorylation site in P-glycoprotein (residues 656-666)(13) . Although P7 does inhibit the histone kinase activity of PKC, it is weaker than P1, P3, and P5 in this regard, and, at concentrations 10 µM, P7 actually stimulates the histone kinase reaction (Table 1). In a previous report, we showed that the mechanism of PKC inhibition by several N-myristoylated peptide substrate analogs of PKC, including P1 and P5, entailed binding interactions of the inhibitor peptides with the active site of PKC and with phospholipid vesicles(27) . In control experiments that monitored the absorbance of the Tyr-containing P1 analog NmYARKGALRQ, we have found that the phospholipid-interacting peptide does not bind to plastic-, glass-, or collagen-coated surfaces (data not shown).

P1 has been shown to inhibit phosphorylation of the PKC substrate MARCKS in intact rat fibroblasts(26) , and the truncated P5 analog N-myristoyl-KRTLR antagonizes PKC-requiring pathways in T-lymphocytes(31, 32) . The demonstrated ability of PKC-inhibitory N-myristoylated peptide substrate analogs to inhibit PKC activity in nonpermeabilized mammalian cells (26, 31, 32) provides indirect evidence that the peptides enter mammalian cells, which is consistent with their membrane-active nature(27) . P5 partially reverses adriamycin resistance in murine fibrosarcoma cells(33) . Specific activation of PKC is correlated with enhancement of MDR in tumor cells, and inhibition of the enzyme is often associated with partial reversal of MDR(10, 11) . We hypothesized that P1 and related PKC-inhibitory N-myristoylated peptides might antagonize MDR in human breast cancer MCF7-MDR cells.

Fig. 1shows the effects of the peptides P1-P6 on the intracellular accumulation of [C]ADR and [H]VLB, which are P-glycoprotein substrates(1, 2, 10) , and [H] 5FU, which is not a P-glycoprotein substrate(1, 2, 10) , in MCF7-MDR cells. All assays of drug accumulation in MCF7-MDR and MCF7-WT cells described in this report were done under conditions where cell viability was >95% at the end of the assay period, according to measurements of trypan blue exclusion. The N-myristoylated peptides P1, P3, and P5 markedly and significantly increased the accumulation of [C]ADR (Fig. 1A) and [H]VLB (Fig. 1B) in the MCF7-MDR cells, whereas the nonmyristoylated peptides P2, P4, and P6 had no effect on their accumulation (Fig. 1, A and B). The N-myristoylated peptides enhanced drug accumulation in the MCF7-MDR cells from 3-9-fold, which is comparable with the enhancement of drug accumulation that was achieved by the potent MDR reversal agent verapamil (VP) (Fig. 1, A and B). For comparison, the levels of intracellular [C]ADR and [H]VLB accumulation in MCF7-WT were respectively 5- and 10-fold higher than those in MCF7-MDR. The enhancement of drug uptake was most pronounced when the N-myristoylated peptide concentration was 100 µM. However, at a concentration of 50 µM, P1 and P3 each significantly enhanced [C]ADR uptake, and P1 and P5 significantly enhanced [H]VLB uptake in the MCF7-MDR cells (Fig. 1, A and B). In parallel experiments, we found that like VP, P1-P6 did not significantly affect [H]5FU accumulation in MCF7-MDR cells (Fig. 1C). This demonstrates a degree of specificity in the effects of the N-myristoylated peptides on drug accumulation in MCF7-MDR cells. [C]ADR and [H]VLB accumulation in MCF7-MDR cells were enhanced by less than 5% by myristic acid (25-100 µM) and by N,N-dimethylmyristamide (25-100 µM), which is a nonpeptidic amide of myristic acid. Thus, specificity is also indicated by the lack of effect of the acyl head group of the peptides on [C]ADR and [H]VLB accumulation. In addition, P7 (25-100 µM) failed to enhance [C]ADR and [H]VLB accumulation in the cells, indicating that not all N-myristoylated cationic peptides can enhance the retention of these cytotoxic drugs in MCF7-MDR cells.

Figure 1: Effects of synthetic peptide substrate analogs of PKC on cytotoxic drug accumulation in MCF7-MDR cells. Effects of the N-myristoylated peptides P1, P3, and P5 and the corresponding nonmyristoylated peptides P2, P4, and P6 at concentrations of 25, 50, and 100 µM on drug accumulation in MCF7-MDR cells are shown. Drug accumulation was assayed as described under ``Materials and Methods.'' Under each experimental condition examined in A-C, >95% cell viability was observed at the end of the drug accumulation period by measurements of trypan blue exclusion. Each experimental value shown is the average value from three experiments that were done in triplicate. Values that differ significantly from the untreated control are identified by asterisks.**, p < 0.015; *, p < 0.05. A, [C]ADR accumulation in MCF7-MDR cells is shown. % of Control, the amount of [C]ADR accumulation observed in the presence of the indicated peptide expressed as a percentage of [C]ADR accumulation observed in untreated control (CTRL) MCF7-MDR cells. Treatment with 10 µM VP served as a positive control. The [C]ADR concentration was 0.1 µM, and accumulation was measured after a 2-h incubation period. Untreated cells retained 2.2 ± 0.2 pmol [C]ADR/10 cells. B, [H]VLB accumulation in MCF7-MDR cells is shown. Assays were done with 10 nM [H]VLB, and accumulation was measured after incubating the cells with drugs for 30 min. Untreated MCF7-MDR cells retained 0.066 ± 0.005 pmol [H]VLB/10 cells. For other details, see the description of A. C, [H]5FU accumulation in MCF7-MDR cells is shown. Where indicated, peptides (P1-P6) were present at 100 µM; [H]5FU was at 10 nM. Drug accumulation was measured after 30 min and 2 h, as shown. [H]5FU accumulation in control (CTRL) cells was 0.058 ± 0.002 (30 min) and 0.058 ± 0.004 pmol [H] 5FU/10 cells (2 h). For other details, see the description of A.

Table 2shows that P1-P6 increased [C]ADR and [H]VLB accumulation in the drug-sensitive cell line MCF7-WT only marginally (<1.8-fold) (P2, P3, P4) or not at all (P1, P5, P6), and they had no detectable effect on [H]5FU accumulation in the cells. Of the N-myristoylated peptides, only P3 detectably increased [C]ADR and [H]VLB accumulation in the MCF7-WT cells (Table 2). Thus, the marked enhancement of [C]ADR and [H]VLB uptake achieved by the N-myristoylated peptides P1, P3, and P5 but not by their nonmyristoylated counterparts (Fig. 1) were specific to the MDR subline.

Having established that the N-myristoylated peptides significantly increase [C]ADR and [H]VLB accumulation in MCF7-MDR cells at 2 h and 30 min, respectively (Fig. 1), we next examined the effects of P1-P6 on the accumulation of [C]ADR and [H]VLB in MCF7-MDR cells over a 6-h time course. In these experiments, cells were pretreated with peptide for 30 min, and the time course was initiated by the addition of drug, as described under ``Materials and Methods.'' The enhancement of drug accumulation by the N-myristoylated peptides (P1, P3, and P5) was observed within 5 and 15 min, respectively, for [H]VLB (Fig. 2, C and D) and [C]ADR (Fig. 2, A and B). The enhancement of [C]ADR accumulation was sustained at a maximal or nearly maximal level over a 6-h time course in each case, as was the enhancement of [H]VLB accumulation by P1 (Fig. 2, A-C), but the effects of P3 and P5 on [H]VLB accumulation peaked within 2 h and then gradually declined throughout the remainder of the time course (Fig. 2D). No enhancement of [C]ADR or [H]VLB accumulation was observed with the nonmyristoylated peptides (P2, P4, and P6) (Fig. 2). The relatively slow uptake of [C]ADR induced by the N-myristoylated peptides allowed estimation of the effects of the peptides on the [C]ADR efflux rate of MCF7-MDR cells by measuring the net efflux of [C]ADR from preloaded cells. Across a 30-min time course, a decline in the net [C]ADR efflux rate was observed in MCF7-MDR cells with each N-myristoylated peptide (100 µM P1, P3, and P5), and the mean value of the decline achieved by the peptides was 13 ± 4%, which was comparable with the decline of 19 ± 2% observed with 10 µM verapamil.

Figure 2: Effects of synthetic peptide substrate analogs of PKC on the kinetics of net drug uptake in MCF7-MDR cells. Intracellular accumulation of [C]ADR (A and B) and [H]VLB (C and D) was measured in MCF7-MDR cells at the indicated time intervals. Where specified, assays were done in the presence of 100 µM peptide (A and C, P1 and P2; B and D, P3, P4, P5, and P6) or 10 µM VP (A and C). In these experiments, cell viability was >95% according to trypan blue exclusion measurements. For definition of % of Control, assay conditions, and other experimental details, see the legend to Fig. 1and ``Materials and Methods.'' Each experimental value represents an average of triplicate determinations, and the results shown were determined to be reproducible in a duplicate experiment.

At a concentration of 50 µM, the N-myristoylated pseudosubstrate peptide P1 significantly enhanced [C]ADR and [H]VLB accumulation in the MCF7-MDR cells, but in each case the degree of enhancement was very modest when compared with 10 µM VP (Fig. 1). Likewise, at concentrations of 2.5 µM, VP had little effect on [C]ADR accumulation (Table 3). In an attempt to demonstrate efficacy of P1 at concentrations of 50 µM, we measured the enhancement of [C]ADR uptake in MCF7-MDR cells by combinations of P1 (50 µM) and VP (2.5 µM). We found that 2.5 µM VP in combination with 25 µM P1 enhanced [C]ADR accumulation in MCF7-MDR cells approximately as effectively as 10 µM VP, and the degree of enhancement was significantly greater than that achieved by either 25 µM P1 or 2.5 µM VP alone (Table 3). We observed a similar but more marked statistically significant effect by 50 µM P1 in combination with 2.5 µM VP (Table 3). These results demonstrate that the effects of P1 and VP on drug accumulation in MCF7-MDR cells are additive, and they show that P1 can modulate drug accumulation in the MDR breast cancer cells at concentrations as low as 25 µM. (5 µM P1 was ineffective in combination with VP in enhancing [C]ADR accumulation) (data not shown).

The restoration of [C]ADR and [H]VLB accumulation by the N-myristoylated pseudosubstrate peptide P1 in MCF7-MDR cells suggested that the peptide might also sensitize MCF7-MDR cells to the cytotoxic drugs. To test this, we measured the effect of a 1-h exposure to P1 on MCF7-MDR cell growth using a 96-h assay system and found that under these conditions, P1 was not growth-inhibitory (Fig. 3). Thus, P1 could be tested for MDR reversal activity in this system under conditions where it potently stimulated drug accumulation in MCF7-MDR cells (Fig. 2, A and C). Under these conditions, P1 significantly reduced the ADR concentration required for 50% MCF7-MDR cell growth inhibition (IC) approximately 2-fold, from a value of 26.33 ± 0.40 µg/ml to 14.11 ± 0.41 µg/ml (p < 0.001, n = 6). Representative results obtained in one experiment are shown in Fig. 3A. Similarly, the IC of VLB was reduced by P1 approximately 2-fold in MCF7-MDR cells, from 2.56 ± 0.32 µg/ml to 1.18 ± 0.08 µg/ml VLB (p < 0.02, n = 5); representative data are shown in Fig. 3B. In contrast, P1 was without effect on 5FU cytotoxicity in MCF7-MDR cells. Fig. 3C shows that the percentage of MCF7-MDR cell growth inhibition achieved by 5FU was approximately the same in the presence (open circles) and in the absence of P1 (closed circles); the results shown are an average of three experiments. Taken together with the effects of P1 on drug accumulation in MCF7-MDR cells ( Fig. 1and Fig. 2), these results provide evidence that P1-mediated chemosensitization of MCF7-MDR cells involves inhibition of P-glycoprotein-mediated drug transport.

Figure 3: Chemosensitization of MCF7-MDR cells by the N-myristoylated PKC- pseudosubstrate peptide P1. Effects of 100 µM P1 on the growth-inhibitory activity of ADR (A), VLB (B), and 5FU (C) in MCF7-MDR cells are shown. In these experiments, cells were treated with P1 for 1 h followed by a 96-h exposure to the cytotoxic drug. Under these conditions, the direct growth-inhibitory effects of P1 were negligible. For other experimental details, see ``Materials and Methods.'' In A and B, results of a single experiment are shown; each point represents an average of eight determinations. In C, the data shown are an average of three experiments, in which each data point was an average of eight determinations.

We next tested whether restoration of drug accumulation in MCF7-MDR cells by P1 and related N-myristoylated PKC-inhibitory peptides was associated with inhibition of P-glycoprotein phosphorylation. Fig. 4A shows that exposure of MCF7-MDR cells to P1 (25-100 µM) during a 3-h P-labeling period was associated with a concentration-dependent inhibition of P-glycoprotein phosphorylation; maximal inhibition was achieved with 100 µM P1. Similarly, P3 and P5 inhibited P-glycoprotein phosphorylation in MCF7-MDR cells, although P5 was considerably less effective than either P1 or P3 in this respect (Table 4). In contrast, the effects of the nonmyristoylated peptides P2, P4, P6, and the N-myristoylated peptide P7 on P-glycoprotein phosphorylation in MCF7-MDR cells were negligible (Table 4). In control experiments, we determined by immunoblot analysis of P-glycoprotein with C219 mAb that P1, P3, and P5 had only minor or negligible effects on P-glycoprotein expression under the conditions of the P-glycoprotein phosphorylation experiments (Fig. 4B; Table 4).

Figure 4: Inhibition of P-glycoprotein phosphorylation in MCF7-MDR cells by the N-myristoylated PKC- pseudosubstrate peptide P1. A, effects of P1 on P-glycoprotein phosphorylation in MCF7-MDR cells were determined by exposing the cells to P1 during a 3-h [P]P-labeling period and recovering P-labeled P-glycoprotein from lysates of P1-treated cells by immunoprecipitation with the mAb C219. The immunoprecipitated protein was subjected to SDS-PAGE analysis followed by autoradiography of gels, as described under ``Materials and Methods.'' MCF7-WT (lane A), MCF7-MDR treated with 100 µM P1 (lane B), 50 µM P1 (lane C), 25 µM P1 (lane D), and 0 µM P1 (lane E) are shown. P-glycoprotein is the 160-kDa band. B, the effect of 100 µM P1 on P-glycoprotein expression in MCF7-MDR cells was measured under the conditions of the P-glycoprotein phosphorylation experiments by immunoblot analysis of cell lysates with the mAb C219 (500 ng/ml), as described under ``Materials and Methods.'' Immunoblots corresponding to untreated (lane 1) and P1-treated MCF7-MDR cells (lane 2) (25 µg of protein/lane) are shown. P-glycoprotein is the band migrating at 160 kDa.

To determine whether the PKC-inhibitory effects of the N-myristoylated peptides in MCF7-MDR cells were restricted to integral membrane protein PKC substrates such as P-glycoprotein, we examined the phosphorylation state of the PKC substrate Raf-1 kinase (34) , which shuttles between the cytoplasmic compartment and the plasma membrane of mammalian cells(35) . Raf-1 kinase has been implicated in MDR(36) , and it plays a pivotal role in PKC--mediated signal transduction(34) . Activation of cellular PKC- triggers a protein kinase cascade that begins with PKC--catalyzed phosphorylation and activation of Raf-1 kinase and ultimately results in the phosphorylation of nuclear proteins(37) . Raf-1 kinase phosphorylation was analyzed by immunoprecipitation of the protein from lysates of P-labeled cells with a Raf-1 kinase-specific mAb. We found that the phosphorylation of Raf-1 kinase was at least 10 times greater in the MCF7-MDR line compared with the drug-sensitive line MCF7-WT (Fig. 5A, lanes F and G), when the phosphorylation data in Fig. 5A were subjected to densitometric analysis and then normalized for the approximately 2-fold increase in Raf-1 kinase expression that we observed in the MDR line by Western analysis (Fig. 5B). This increase in the phosphorylation of the PKC- substrate Raf-1 kinase in MCF7-MDR is consistent with the reported 30-fold overexpression of PKC- in the cells(18) . As in the case of P-glycoprotein phosphorylation, 100 µM P1, P3, and P5 each potently inhibited Raf-1 kinase phosphorylation in the MCF7-MDR cells, whereas 100 µM P7 was noninhibitory (Fig. 5A, lanes B-E). In contrast, the peptides had little or no effect on Raf-1 kinase expression in MCF7-MDR cells, according to immunoblot analysis of cell lysates (Fig. 5B). Densitometric analysis revealed that 100 µM P3 and P5 each inhibited Raf-1 kinase phosphorylation in MCF7-MDR cells by more than 95% (Fig. 5A, lanes C and D), and 200 µM P1 achieved a similar degree of inhibition (Fig. 5A, lane A). The inhibition of Raf-1 kinase phosphorylation achieved by 100 µM P1 was approximately 80% (Fig. 5A, lane B).

Figure 5: Inhibition of Raf-1 kinase phosphorylation in MCF7-MDR cells by N-myristoylated synthetic peptide substrate analogs of PKC. A, MCF7-WT or MCF7-MDR cells were labeled with P for 3 h prior to detergent lysis of cells and recovery of Raf-1 kinase by immunoprecipitation with a Raf-1 kinase mAb as described under ``Materials and Methods.'' Where indicated, MCF7-MDR cells were treated with N-myristoylated synthetic peptide substrate analogs of PKC for the duration of the 3-h labeling period. Immunoprecipitated Raf-1 kinase was analyzed by SDS-PAGE followed by autoradiography. The same exposure period was used for each lane. MCF7-MDR cells were treated with 200 µM P1 (lane A), 100 µM P1 (lane B), 100 µM P3 (lane C), 100 µM P5 (lane D), or 100 µM P7 (lane E). In control lanes, untreated MCF7-WT (lane F) and MCF7-MDR (lane G) cells are shown. Raf-1 kinase migrated as a 72-kDa band. B, immunoblot analysis of Raf-1 kinase expression in MCF7 cell lysates (50 µg of cell lysate protein/lane) using 10 µg/ml Raf-1 kinase monoclonal antibody as the primary antibody is shown in the upper panel; the lower panel shows the results of a control immunoblot analysis done in the absence of primary antibody. The arrows indicate the positions of MCF7-MDR Raf-1 kinase (72 kDa); MCF7-WT Raf-1 kinase migrated slightly more rapidly (68 kDa). Cells were treated with peptides as described for A. Lane 1, MCF7-WT; lane 2, MCF7-MDR; lane 3, P1-treated MCF7-MDR; lane 4, P3-treated MCF7-MDR; lane 5, P5-treated MCF7-MDR; lane 6, P7-treated MCF7-MDR. Peptides were present at 100 µM. The results shown in A and B were reproducible in separate experiments.

PKC--catalyzed Raf-1 kinase phosphorylation is preceded by PKC- activation and autophosphorylation(38, 39) . To test whether the alterations in Raf-1 kinase phosphorylation shown in Fig. 5A were reflective of changes in PKC- activity, we analyzed the MCF7-WT and MDR cells for comparable changes in PKC- phosphorylation. Like Raf-1 kinase, PKC- is localized in the plasma membrane and the cytoplasmic compartment of mammalian cells(17, 40) . Following a 3-h labeling period, a major 82-kDa radiolabeled band corresponding to phosphorylated PKC- was detected in P-labeled MCF7-MDR but not in MCF7-WT by immunoprecipitation of the protein from the cell lysates with a PKC--specific mAb (Fig. 6A, lanes A and F). Similarly, PKC- was readily detected in an MCF7-MDR cell lysate but not in an MCF7-WT cell lysate by immunoblot analysis (Fig. 6B, lanes 1 and 2). Densitometric analysis of Fig. 6A showed that as in the case of Raf-1 kinase phosphorylation, PKC- phosphorylation was inhibited in MCF7-MDR cells >95% by 100 µM P3 (lane C) and 100 µM P5 (lane D) and approximately 80% by 100 µM P1 (lane B); 100 µM P7 affected <25% inhibition of PKC- phosphorylation (lane E). The peptides had little or no effect on the expression of PKC- in MCF7-MDR cells under these experimental conditions according to immunoblot analysis of cell lysates (Fig. 6B). At 200 µM, P1 inhibited PKC- phosphorylation in MCF7-MDR cells >95%, but P7 still achieved <25% inhibition (data not shown). Thus, the N-myristoylated peptides P1, P3, and P5 potently inhibited the phosphorylation of three PKC- substrates (P-glycoprotein, Raf-1 kinase, and PKC-) in MCF7-MDR cells under conditions where the peptides restored intracellular drug accumulation, whereas the N-myristoylated peptide P7 inhibited the phosphorylation of these PKC- substrates very weakly or not at all and was without effect on the uptake of cytotoxic drugs in the MDR breast cancer cells. In most cases, the peptide concentration required for potent induction of drug uptake by P1, P3, and P5 (100 µM) (Fig. 1) caused 80% inhibition of the phosphorylation of P-glycoprotein, Raf-1-kinase, and PKC- in the MCF7-MDR cells (Fig. 4Fig. 5Fig. 6; Table 4), suggesting that nearly complete inhibition of PKC- catalysis may be required for substantial reversal of MDR by the peptides.

Figure 6: Inhibition of PKC- phosphorylation in MCF7-MDR cells by N-myristoylated synthetic peptide-substrate analogs of PKC. A, MCF7-WT or MCF7-MDR cells were incubated with [P]P in phosphate-free medium for 3 h as described under ``Materials and Methods.'' Where indicated, MCF7-MDR cells were treated with synthetic peptides (P1, P3, P5, and P7) for the duration of the labeling period. P-labeled cells were lysed with detergent, and PKC- was immunoprecipitated from the cell lysates, as described under ``Materials and Methods.'' Immunoprecipitated PKC- was detected by SDS-PAGE and autoradiography of the gel; the autoradiogram was subjected to densitometric analysis. Results are shown for MCF7-WT (lane A), MCF7-MDR treated with 100 µM P1 (lane B), 100 µM P3 (lane C), 100 µM P5 (lane D), 100 µM P7 (lane E), and untreated MCF7-MDR (lane F). PKC- is the radiolabeled band migrating at 82 kDa. B, immunoblot analysis of PKC- expression in MCF7 cell lysates (50 µg of protein/lane) using 1 µg/ml PKC- monoclonal antibody as the primary antibody (12) is shown in the upper panel; a control analysis done in the absence of the primary antibody is shown in the lower panel. The arrows indicate the positions of PKC- (82 kDa). Cells were treated with peptides as described for A. Lane 1, MCF7-WT; lane 2, MCF7-MDR; lane 3, P1-treated MCF7-MDR; lane 4, P3-treated MCF7-MDR; lane 5, P5-treated MCF7-MDR; lane 6, P7-treated MCF7-MDR. Peptides were present at 100 µM. The results shown in A and B were reproducible in separate experiments.

Prenylcysteine methyl esters and several cyclic peptides have been shown to compete for drug-binding sites on P-glycoprotein in studies of photoaffinity labeling of the transporter with [H]azidopine(41, 42) , and several hydrophobic linear peptides have been reported to serve as P-glycoprotein substrates (42, 43, 44, 45) . In contrast, the amphiphilic linear peptides melittin and alamethicin do not interact with P-glycoprotein(42) . To test whether the mechanism of MDR reversal by the amphiphilic peptide P1 could involve direct interactions between P1 and drug-binding sites of P-glycoprotein in addition to inhibition of P-glycoprotein phosphorylation, we analyzed the effects of P1 and related peptides on the photoaffinity labeling of P-glycoprotein with [H]azidopine in MCF7-MDR cells. Results of photoaffinity labeling experiments done in whole MCF7 cells are shown in Fig. 7. A single prominent [H]azidopine-labeled band that corresponded to P-glycoprotein (160 kDa) was observed in the lane corresponding to untreated MCF7-MDR cells (lane B); the absence of this band in the MCF7-WT sample (lane A) confirmed its identity as P-glycoprotein. Exposure of the MCF7-MDR cells to vinblastine achieved about 50% inhibition of the labeling of P-glycoprotein according to densitometric analysis (lane C). Exposure to 100 µM P1, P3, and P5 (lanes D-F) had no inhibitory effect on photoaffinity labeling of P-glycoprotein in MCF7-MDR cells. In fact, the peptides actually enhanced the labeling of P-glycoprotein by [H]azidopine.

Figure 7: Effects of N-myristoylated synthetic peptide substrate analogs of PKC on photoaffinity labeling of P-glycoprotein by [H] azidopine in MCF7-MDR cells. Photoaffinity labeling of P-glycoprotein in MCF7-MDR cells was done following preincubation of the cells with [H]azidopine and the peptide under investigation in the dark, as described under ``Materials and Methods.'' Peptides were employed at a concentration of 100 µM. Photoaffinity labeling was followed by extraction and immunoprecipitation of P-glycoprotein and SDS-PAGE analysis. [H]Azidopine-labeled P-glycoprotein, which migrated as a 160-kDa band, was quantitated by autoradiography and computerized densitometry. Samples were untreated MCF7-WT (lane A), untreated MCF7-MDR (lane B), MCF7-MDR treated with 30 µM VLB (lane C), 100 µM P1 (lane D), 100 µM P3 (lane E), and 100 µM P5 (lane F).

Comparisons of the chemosensitivities of MCF7-MDR and MCF7-WT cells to cytotoxic drugs have shown that the relative resistances of MCF7-MDR cells to the P-glycoprotein substrates ADR and VLB are, respectively, 610- and 360-fold(18) . As a test of whether P1 could serve as a P-glycoprotein substrate in MCF7-MDR cells, we analyzed the MCF7-MDR cells for cross-resistance to P1 by comparing the growth-inhibitory activity of P1 and related peptides against MCF7-WT and MCF7-MDR cells. In these experiments, cells were exposed to the N-myristoylated peptides during the entire growth inhibition assay period (96 h; the 1-h exposure period employed in Fig. 3was not used because it would have required very high peptide concentrations to achieve >50% cell growth inhibition). Although statistically significant cross-resistance was observed in MCF7-MDR cells with each peptide, the very modest degree of cross-resistance to the pseudosubstrate peptides P1 and P3 (<1.5-fold) (Table 5) provided evidence that P1 and P3 did not serve effectively as P-glycoprotein substrates in the cells. In contrast, the cross-resistance of the cells to P5, which contains a sequence that corresponds to a PKC phosphorylation site in the EGF receptor, was pronounced (10-fold) (Table 5), providing evidence that P5 may be transported by P-glycoprotein. Ideally, an MDR reversal agent should be equipotent against drug-sensitive and MDR cancer cells in its direct growth-inhibitory effects, because cross-resistance of the MDR cells to the agent could necessitate its use at concentrations that are directly toxic to nontransformed cells to accomplish reversal of MDR in cancer cells in vivo. Thus, the minor degree of cross-resistance of the MCF7-MDR cells to the N-myristoylated pseudosubstrate peptides P1 and P3 (Table 5) further indicates the potential value of the pseudosubstrate peptides as MDR reversal agents. In contrast, the pronounced cross-resistance of the MCF7-MDR cells to the EGF receptor-related peptide P5 indicates that it is not appropriate for MDR reversal.

DISCUSSION

Previous reports have shown that the isozyme PKC- is selectively overexpressed in human breast cancer MCF7-MDR cells(18) , and artificial overexpression of PKC- in MCF7 constructs that overexpress P-glycoprotein increases the drug resistance of the cells in association with increased P-glycoprotein phosphorylation(19) , providing evidence that PKC--catalyzed P-glycoprotein phosphorylation may contribute to MDR in MCF7 cells. In this report, we demonstrate that an N-myristoylated PKC- pseudosubstrate peptide, N-myristoyl-FARKGALRQ(P1), partially reverses drug resistance in MCF7-MDR by a novel mechanism that involves PKC- inhibition. P1 induced cytotoxic drug accumulation in MCF7-MDR cells just as effectively as the potent MDR reversal agent verapamil (Fig. 1) in association with potent inhibition of P-glycoprotein phosphorylation (Fig. 4). P1 also inhibited the phosphorylation of two other PKC- substrates in MCF7-MDR cells, Raf-1 kinase (Fig. 5), and PKC- (Fig. 6) under these conditions. Thus, induction of drug accumulation by P1 was associated with PKC- inhibition in MCF7-MDR cells. Based on the evidence described above (18, 19) that PKC--catalyzed P-glycoprotein phosphorylation may be a contributing factor in the MDR phenotype of MCF7-MDR cells, it is evident that the mechanism of MDR reversal by P1 most likely involves inhibition of P-glycoprotein phosphorylation.

The mechanism of P1-mediated MDR reversal clearly does not involve competitive binding at [H]azidopine binding sites on P-glycoprotein (Fig. 7). This distinguishes P1 from MDR reversal agents such as PKC-inhibitory staurosporines(47, 48) , verapamil, and cyclosporin A, which are highly effective MDR reversal agents in vitro but cannot be used to reverse MDR in vivo due to severe toxic effects at therapeutic concentrations(24, 47) . It is also evident that MDR reversal by P1 does not involve altered P-glycoprotein expression and that it is not compromised by cross-resistance in the MDR cells (Fig. 4B, Table 5). It should be noted that modulation of the ATPase activity of isolated P-glycoprotein and the [H]VLB binding activity of P-glycoprotein-containing membrane vesicles by phospholipid-interacting peptides, such as melittin (42) and the N-myristoylated peptides described here(27) , cannot be used to characterize interactions between the peptides and P-glycoprotein because of the pronounced nonspecific effects of phospholipid-interacting peptides in these assay systems (42) . It is also worthwhile to note that because treatment of breast cancer patients with tamoxifen, which is PKC-inhibitory at therapeutic concentrations(49) , is associated with little toxicity, it appears that PKC-inhibitory MDR reversal agents such as P1 could potentially give rise to a new generation of MDR reversal agents that are associated with acceptably low toxicity.

The inability of P1 to antagonize [H]azidopine labeling of P-glycoprotein in MCF7-MDR cells and the lack of cross-resistance of MCF7-MDR cells to P1 suggest that P1 is not a P-glycoprotein substrate. However, these results do not exclude the possibility that linear myristoylated peptides such as P1 may interact with a P-glycoprotein site that is nonoverlapping and distinct from the azidopine binding site. In fact, the enhancement of [H]azidopine labeling of P-glycoprotein by P1 is suggestive of such interactions. The enhanced labeling affected by P1 cannot be explained simply by the inhibitory activity of P1 against P-glycoprotein phosphorylation, because PKC--catalyzed P-glycoprotein phosphorylation increases azidopine-labeling of the pump (46) . Nor can it be explained by the amphiphilicity of P1, because other linear amphiphilic peptides do not enhance azidopine labeling of P-glycoprotein(42) .

In this study, we compared the effects of P1 and other N-myristoylated peptide-substrate analogs of PKC in MCF7-MDR cells. In general, the ability of the N-myristoylated peptide-substrate analogs to inhibit the phosphorylation of endogenous PKC- substrates (P-glycoprotein, Raf-1 kinase, and PKC-) in MCF7-MDR cells correlated with restoration of drug uptake in the cells by the peptides, i.e. P1, P3, and P5 were active, whereas P7 was inactive. Discrepancies within this general trend may be due to the pronounced cross-resistance of the MCF7-MDR cells to P5, effects of the N-myristoylated peptides on P-glycoprotein in addition to inhibition of the phosphorylation of the pump (these putative effects are inferred from the enhancement of photoaffinity labeling of P-glycoprotein by the peptides as discussed above), differences among the amphiphilic peptides in their interactions with cell membranes, etc. Furthermore, because P1 and P3 contain PKC- pseudophosphorylation sequences(26, 27) , they are likely to be recognized not only by PKC- but also by other proteins that interact with naturally occurring PKC- phosphorylation sites, e.g. protein phosphatases. It is also important to note that because of the overlapping substrate specificities of PKC isozymes(50) , P1 and P3 may also antagonize the function of several other PKC isozymes in MCF7-MDR cells, and these inhibitory effects may contribute to their MDR reversal activity. Finally, complexity is also introduced by the existence of multiple mechanisms of drug resistance in drug-selected MDR cancer cells that overexpress P-glycoprotein, such as MCF7-MDR. Contributing factors to MDR can include multidrug resistance protein (MRP), glutathione S-transferase, topoisomerases, etc.(25) , and some of these drug resistance mechanisms might also be affected by the peptides.

Studies of the inhibition of purified PKC by N-myristoylated peptide substrate analogs demonstrated that the N-myristoylated peptides interacted with the phospholipid cofactor, whereas their nonmyristoylated counterparts did not(27) . The membrane-active nature of the N-myristoylated peptides most likely accounts for their ability to access cellular PKC(26, 31, 33) . As the phospholipid cofactor concentration is increased in the PKC assay system, the inhibitory potency of the N-myristoylated peptides declines(27) , providing evidence that their inhibitory effects are subject to surface dilution. This may account for the sharp increase in the potencies of P1, P3, and P5 as inducers of drug uptake in MCF7-MDR cells when the bulk peptide concentration was increased from 50 to 100 µM.

Bioactive peptides such as P1 are subject to proteolytic degradation, which often limits their potency. This may explain the superior potency of P1 in inducing drug uptake in short-term assays (6 h) compared with its potency in reversing MDR over a 96-h time course. In some cases, this problem can be overcome by designing retro-inverso analogs of the peptides. In a retro-inverso analog, the sequence of the parent peptide is reversed, and the residues are replaced by the corresponding D-enantiomers(51) . The side chain surfaces of parent and retro-inverso peptides are similar or identical, but the topologies of their backbones differ(51) . Thus, superior bioactivity can be achieved by retro-inverso analogs, when the side chain surface prevails in the interaction of the bioactive peptide with its target(52) . A retro-inverso analog of a PKC- pseudosubstrate peptide has been shown to potently inhibit phosphorylation of a synthetic peptide-substrate by PKC(53) . Efforts are now underway to design retro-inverso analogs of P1 and related N-myristoylated peptides that are superior to the parent peptides in the reversal of MDR in human breast cancer cells. In a recent report, computer-based algorithms were successfully used to predict MDR reversal activity based on structural features of compounds entered into the data base (54) ; this type of strategy may also be useful for designing peptidic or peptidomimetic P1 analogs with optimized MDR reversal activity.

FOOTNOTES

*

This work was supported by Robert A. Welch Foundation Grant G-1141, National Cancer Institute Grant CA-52460, and an award from The Elsa U. Pardee Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Dept. of Cell Biology, Box 173, UTMD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030.

()

The abbreviations used are: MDR, multidrug-resistant or multidrug resistance; ADR, Adriamycin; 5FU, 5-fluorouracil; PKC, protein kinase C; VLB, vinblastine; VP, verapamil; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; mAb, monoclonal antibody; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

ACKNOWLEDGEMENTS

REFERENCES

1. Gottesman, M. M., and Pastan, I. (1993) Annu. Rev. Biochem. 62, 385-427 [CrossRef][Medline] [Order article via Infotrieve]
2. Endicott, J. A., and Ling, V. (1989) Annu. Rev. Biochem. 58, 137-171 [CrossRef][Medline] [Order article via Infotrieve]
3. Goldstein, L. J., Galski, H., Fojo, A., Willingham, M., Lai, S.-L., Gazdar, A., Pirker, R., Green, A., Crist, W., Brodeur, G. M., Lieber, M., Cossman, J., Gottesman, M. M., and Pastan, I. (1989) J. Natl. Cancer Inst. 81, 116-124 [Abstract/Free Full Text]
4. Weinstein, R. S., Jakate, S. M., Dominguez, J. M., Lebovitz., M. D., Koukoulis, G. K., Kuszak, J. R., Klusens, L. F., Grogan, T. M., Saclarides, T. J., Roninson, I. B., and Coon, J. S. (1991) Cancer Res. 51, 2720-2726 [Abstract/Free Full Text]
5. Asaoka, Y., Nakamura, S., Yoshida, K., and Nishizuka, Y. (1992) Trends Biochem. Sci. 17, 414-417 [CrossRef][Medline] [Order article via Infotrieve]
6. Ferguson, P. J., and Cheng, Y. (1987) Cancer Res. 47, 433-441 [Abstract/Free Full Text]
7. Fine, R. L., Patel, J., and Chabner, B. A. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 582-586 [Abstract/Free Full Text]
8. O'Brian, C. A., Fan, D., Ward, N. E., Dong, Z., Iwamoto, L., Gupta, K . P., Earnest, L. E., and Fidler, I. J. (1991) Biochem. Pharmacol. 41, 797-806 [CrossRef][Medline] [Order article via Infotrieve]
9. Chambers, T. C., McAvoy, E. M., Jacobs, J. W., and Eilon, G. (1990) J. Biol. Chem. 265, 7679-7686 [Abstract/Free Full Text]
10. Dong, Z., Ward, N. E., Fan, D., Gupta, K. P., and O'Brian, C. A. (1991) Mol. Pharmacol. 39, 563-569 [Abstract]
11. Chambers, T. C., Zheng, B., and Kuo, J. F. (1992) Mol. Pharmacol. 41, 1008-1015 [Abstract]
12. Gravitt, K. R., Ward, N. E., Fan, D., Skibber, J. M., Levin, B., and O'Brian, C. A. (1994) Biochem. Pharmacol. 48, 375-381 [CrossRef][Medline] [Order article via Infotrieve]
13. Chambers, T. C., Pohl, J., Raynor, R. L., and Kuo, J. F. (1993) J. Biol. Chem. 268, 4592-4595 [Abstract/Free Full Text]
14. Orr, G. A., Han, E. K. H., Browne, P. C., Nieves, E., O'Conner, B. M., Yang, C. P. H., and Horwitz, S. B. (1993) J. Biol. Chem. 268, 25054-25062 [Abstract/Free Full Text]
15. O'Brian, C. A., Fan, D., Ward, N. E., Seid, C., and Fidler, I. J. (1989) FEBS Lett. 246, 78-82 [CrossRef][Medline] [Order article via Infotrieve]
16. Posada, J. A., McKeegan, E. M., Worthington, K. F., Morin, M. J., Jaken, S., and Tritton, T. R. (1989) Cancer Commun. 1, 285-292 [Medline] [Order article via Infotrieve]
17. Lee, S. A., Karaszkiewicz, J. W., and Anderson, W. B. (1992) Cancer Res. 52, 3750-3759 [Abstract/Free Full Text]
18. Blobe, G. C., Sachs, C. W., Khan, W. A., Fabbro, D., Stabel, S., Wetsel, W. C., Obeid, L. M., Fine, R. L., and Hannun, Y. A. (1993) J. Biol. Chem. 268, 658-664 [Abstract/Free Full Text]
19. Yu, G., Ahmad, S., Aquino, A., Fairchild, C. R., Trepel, J. B., Ohno, S., Suzuki, K., Tsuruo, T., Cowan, K. H., and Glazer, R. I. (1991) Cancer Commun. 3, 181-189 [Medline] [Order article via Infotrieve]
20. Kang, Y., and Perry, R. R. (1994) Cancer Res. 54, 2952-2958 [Abstract/Free Full Text]
21. Chen, G., Ramachandran, C., and Krishan, A. (1993) Cancer Res. 53, 2544-2547 [Abstract/Free Full Text]
22. Tamai, I., and Safa, A. R. (1991) J. Biol. Chem. 266, 16796-16800 [Abstract/Free Full Text]
23. Kiue, A., Sano, T., Suzuki, K., Inada, H., Okumura, M., Kikuchi, J., Sato, S., Kohno, K., and Kuwano, M. (1990) Cancer Res. 50, 310-317 [Abstract/Free Full Text]
24. Dalton, W., and Sikic, B. I. (1994) J. NIH Res. 6, 54-57
25. O'Brian, C. A., Ward, N. E., Gupta, K. P., and Gravitt, K. R. (1995) in Alternative Mechanisms of Multidrug Resistance in Cancer (Kellen, J., ed) pp. 173-190, Birkhaueser Publishers, New York
26. Eichholtz, T., de Bont, D. B., de Widt, J., Liskamp, R. M., and Ploegh, H. L. (1993) J. Biol. Chem. 268, 1982-1986 [Abstract/Free Full Text]
27. Ward, N. E., and O'Brian, C. A. (1993) Biochemistry 32, 11903-11909 [CrossRef][Medline] [Order article via Infotrieve]
28. O'Brian, C. A., and Ward, N. E. (1990) Biochemistry 29, 4278-4282 [CrossRef][Medline] [Order article via Infotrieve]
29. Bates, S. E., Lee, J. S., Dickstein, B., Spolyar, M., and Fojo, A. T. (1993) Biochemistry 32, 9156-9164 [CrossRef][Medline] [Order article via Infotrieve]
30. Zheng, B., Chambers, T. C., Raynor, R. L., Markham, P. N., Gebel, H. M., Vogler, W. R., and Kuo, J. F. (1994) J. Biol. Chem. 269, 12332-12338 [Abstract/Free Full Text]
31. Ioannides, C. G., Freedman, R. S., Liskamp, R. M., Ward, N. E., and O'Brian, C. A. (1990) Cell Immunol. 131, 242-252 [CrossRef][Medline] [Order article via Infotrieve]
32. Barja, P., Alavi-Nassab, A., Turck, C. W., and Freire-Moar, J. (1994) Cell Immunol. 153, 28-38 [CrossRef][Medline] [Order article via Infotrieve]
33. O'Brian, C. A., Ward, N. E., Liskamp, R. M., de Bont, D. B., Earnest, L. E., van Boom, J. H., and Fan, D. (1991) Invest. New Drugs 9, 169-179 [Medline] [Order article via Infotrieve]
34. Kolch, W., Heidecker, G., Kochs, G., Hummel, R., Vahidi, H., Mischak, H., Finkenzeller, G., Marme, D., and Rapp, U. R. (1993) Nature 364, 249-252 [CrossRef][Medline] [Order article via Infotrieve]
35. Leevers, S. J., Paterson, H. F., and Marshall, C. J. (1994) Nature 369, 411-414 [CrossRef][Medline] [Order article via Infotrieve]
36. Cornwell, M. M., and Smith, D. E. (1993) J. Biol. Chem. 268, 15347-15350 [Abstract/Free Full Text]
37. Egan, S. E., and Weinberg, R. A. (1993) Nature 365, 781-783 [CrossRef][Medline] [Order article via Infotrieve]
38. Huang, K. P., Nakabayashi, H., and Huang, F. L. (1986) Proc. Natl. Acad. Sci. 83, 8535-8539 [Abstract/Free Full Text]
39. Dutil, E. M., Keranen, L. M., DePaoli-Roach, A. A., and Newton, A. C. (1994) J. Biol. Chem. 269, 29359-29362 [Abstract/Free Full Text]
40. Epand, R. M., and Lester, D. S. (1990) Trends Pharmacol. Sci. 11, 317-320 [CrossRef][Medline] [Order article via Infotrieve]
41. Zhang, L., Sachs, C. W., Fine, R. L., and Casey, P. J. (1994) J. Biol. Chem. 269, 15973-15976 [Abstract/Free Full Text]
42. Sharom, F. J., DiDiodato, G., Yu, X., and Ashbourne, K. J. D. (1995) J. Biol. Chem. 270, 10334-10341 [Abstract/Free Full Text]
43. Raymond, M., Gros, P., Whiteway, M., and Thomas, D. Y. (1992) Science 256, 232-234 [Abstract/Free Full Text]
44. Sharma, R. C., Inoue, S., Roitelman, J., Schimke, R. T., and Simoni, R. D. (1992) J. Biol. Chem. 267, 5731-5734 [Abstract/Free Full Text]
45. Sarkadi, B., Muller, M., Homolya, L., Hollo, Z., Seprodi, J., Germann, U. A., Gottesman, M. M., Price, E. M., and Boucher, R. C. (1994) FASEB J. 8, 766-770 [Abstract]
46. Ahmad, S., Safa, A. R., and Glazer, R. I. (1994) Biochemistry 33, 10313-10318 [CrossRef][Medline] [Order article via Infotrieve]
47. Miyamoto, K., Inoko, K., Wakusawa, S., Kajita, S., Hasegawa, T., Takagi, K., and Koyama, M. (1993) Cancer Res. 53, 1555-1559 [Abstract/Free Full Text]
48. Sato, W., Yusa, K., Naito, M., and Tsuruo, T. (1990) Biochem. Biophys. Res. Commun. 173, 1252-1257 [CrossRef][Medline] [Order article via Infotrieve]
49. O'Brian, C. A., Liskamp, R. M., Solomon, D. H., and Weinstein, I. B. (1985) Cancer Res. 45, 2462-2465 [Abstract/Free Full Text]
50. Dekker, L. V., and Parker, P. J. (1994) Trends Biochem. Sci. 19, 73-77 [CrossRef][Medline] [Order article via Infotrieve]
51. Brady, L., and Dodson, G. (1994) Nature 368, 692-693 [CrossRef][Medline] [Order article via Infotrieve]
52. Jameson, B. A., McDonnell, J. M., Marini, J. C., and Korngold, R. (1994) Nature 368, 744-746 [CrossRef][Medline] [Order article via Infotrieve]
53. Ricouart, A., Tartar, A., Sergheraert, C. (1989) Biochem. Biophys. Res. Commun. 165, 1382-1390 [CrossRef][Medline] [Order article via Infotrieve]
54. Klopman, G., Srivastava, S., Kolossvary, I., Epand, R. F., Ahmed, N., and Epand, R. M. (1992) Cancer Res. 52, 4121-4129 [Abstract/Free Full Text]

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